Carcinogenesis is a multi-step process involving the activation of cellular proto-oncogenes and inactivation of tumor suppressor genes. Epithelial cells undergo different stages of phenotypic and genotypic alterations and gradually acquire malignant growth characteristics. In spite of the relatively high frequency of oral cancers, little is known about the molecular events that lead to disease initiation and progression. The goals of our study are to develop an in vitro model of oral carcinogenesis using immortalized human gingival keratinocytes (IHGK) and to characterize the growth and differentiation properties of normal human gingival keratinocytes (NHGK), IHGK and a number of head and neck squamous cell carcinoma (HNSCC) lines and to relate the changes in phenotype with the gene expression profiles. Gene expression profiling of 22 HNSCC lines was also carried out using normal human gingival keratinocytes (NHGK) as a reference. Analysis of the clustered data revealed two distinctive subtypes of gene-expression patterns among the HNSCC lines suggesting a degree of heterogeneity among the gene-expression profiles of HNSCC. There were also a group of genes upregulated or down regulated in HNSCCs with respect to NHGK. The genes upregulated in HNSCC include oncogenes such as ECT2 (epithelial cell transforming sequence 2 oncoprotein), STMN1 (stathmin/oncoprotein 18), DEK oncogene, RACGAP1, growth regulatory factors such as replication factor C, replication protein A3, CKS2 (CDC28 protein kinase2), PCNA (proliferating cell nuclear antigen), CDC20 (CDC20 cell division cycle 20 homolog and keratin 8. The genes commonly downregulated in HNSCC include many keratins, (keratins 1, 2A, 6A, 7, 14, 15, 16, 17, B1 and B3), differentiation markers, (small proline rich protein 3 (SPR3) and 1B (SPR1B), and transglutaminase 3), growth arrest or tumor suppressor gene (GADD45A and FAT), cell surface and extracellular matrix proteins (annexins A1, A2 and A3, laminins alpha and gamma2, integrin beta1, envoplakin, periplakin, corneodesmosin and desmoglein3) and various protease inhibitor proteins (SERPINB1, SERPINB5, SKALP, and SLPI). The in-depth analysis of the gene expression profile of HNSCC should provide potential molecular markers that can be useful for diagnostic and/or therapeutic purposes.